Effects of probiotics in the treatment of alcoholic hepatitis: randomized controlled multicenter study

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1 Effects of probiotics in the treatment of alcoholic hepatitis: randomized controlled multicenter study Lactobacillus subtilis/streptococcus faecium Lactobacillus rhamnosus R0011/acidophilus R0052 Ki Tae Suk, 1 Dong Joon Kim, 1 Moon young Kim, 2 Soon Koo Baik, 2 Young Don Kim, 3 Gab Jin Cheon, 3 Dae Hee Choi, 4 Dong Hoon Shin, 1 Eun Ji Kim, 1 Hak Cheol Kwon, 5 Sung Hun Kim 5

2 Microbiota & Liver Disease Human gut microbiota: complex ecosystem (>>1,000 species, >1kg) Functions: barrier, digestion, and immune system.. Important role in many diseases (IBD, IBS, DM, obesity ) One of the hottest areas in medicine Microbial factor Mediator Clinical implications Small intestinal bacterial overgrowth (SIBO) Multiple NAFLD, ALD Alteration in the composition of microbiota Multiple Obesity, ALD Bacterial translocation LPS, endotoxin SBP, portal hypertension Direct effects of bacterial metabolites Acetaldehyde NAFLD, ALD Nat Rev Gastroenterol Hepatol Dec 20;9(2):72-4 J Hepatol May;58(5): Floch et al. Gastroenterology 1970:59;76

3 Healthy condition Pathologic condition Exacerbate activation of hepatic innate immune system Fibrosis Hepatitis Alcohic liver disease NAFLD Cancer Activation of pro-inflammatory pathway anti-apoptotic pathway anti-viral pathway Hepatology Jan;59(1):328-39

4 Healthy group Fecal Microbial Communities Liver cirrhosis group In patients with liver cirrhosis, the increase of pathogenic bacteria such as Streptococcaceae and the reduction of beneficial bacteria may affect prognosis. Hepatology 2011;54:

5 Alcoholic Liver Disease & Microbiota Alcoholic hepatitis Enterococcus E. coli Lactobacillus reduce endotoxin improve steatohepatitis restore gut flora Hepatology, 2004:39; J Clin Gatroenterol 2005;39: Alcohol Dis, 2000;8:1 4 However, most relevant evidences are from animal studies. Few human studies are available.

6 Hypothesis & Aim Pathophysiological importance of this association is not yet fully understood. Few human data are available in regard to alcoholic liver disease. We evaluated the therapeutic effects of probiotics in patients with alcoholic hepatitis Hepatology Jan;59(1):328-39

7 Design -Multicenter, randomized, clinical trial at 4 university hospitals- Day 0 Day 1 Day 7 Alcoholic hepatitis Admission Randomization Probiotics + Legalon Placebo + Legalon Liver enzymes TNF-a / IL-1b / IL-10 Lipopolysaccharide (LPS) Stool culture (study 1) Stool PCR-DGGE (study 2) Liver enzymes TNF-a / IL-1b / IL-10 LPS Stool culture (study 1) Stool PCR-DGGE (study 2) Stool culture: E. coli & Enterococcus species Colony forming unit (CFU) per gram stool were counted and bacteria were identified by using Vitek II system Stool PCR DGGE:16S rrna V3 Alcohol Dec;42(8): Hepatology, 2004;34:

8 PCR-Denaturing Gradient Gel Electrophoresis (DGGE)

9 Primer sets for bacterial identification/dgge KEY totally conserved conserved variable highly variable >75% variable regions priming sites Bacterial variability map. E. coil 16S rrna gene sequence annotated with bacteria and universal priming sites and variable regions V1-V9.

10 Patients & Methods Alcoholic hepatitis AST/ALT >2 & elevated AST (ALT) level Alcohol >60 g/day (M), >40 g/day (F) during 7 days before screening Last drinks: within 48 hours prior to admission All patients: hospitalization & no alcohol drinking during 7 days. Exclusion: viral hepatitis, autoimmune hepatitis, pancreatitis, delirium tremens, hemochromatosis, Wilson s disease, DILI, cancer, infection need for antibiotics, severe AH, or obesity (BMI >30 kg/m 2 )

11 Study 1 Lactobacillus subtilis/streptococcus faecium Lactowel, 1,500 mg/day Stool culture: CFU per gram stool September 2010 September patients were recruited Randomization Probiotics group (n = 65) Placebo group (n = 65) Excluded because of patients refusal (n = 3), early discharge (n = 1), other (n = 1) Excluded because of patients refusal (n = 3), early discharge (n = 5) Completed full therapy (n = 60) Completed full therapy (n = 57)

12 Study 2, Interim data Lactobacillus rhamnosus R0011/acidophilus R0052 (Lacidophil, 120 mg/day) PCR DGGE, 16S rrna V3 December 2012 September 2014 (Ongoing) 100/150 (66%) patients were recruited Randomization Probiotics group (n = 55) Placebo group (n = 45) Excluded because of patients refusal (n = 3), early discharge (n = 2), Excluded because of patients refusal (n = 3), early discharge (n = 2) Completed full therapy (n = 50) Completed full therapy (n = 40)

13 Study 1 Characteristics of Patients Variables All patients (n = 117) Male (n [%]) 75 (64) Age (years) 52.7 ± 11.3 Presence of L/C (n [%]) 62 (53) Day 1 Day 7 p value Total protein (g/dl) 6.5 ± ± Albumin (g/dl) 3.7 ± ± AST (IU/L) 159 ± ± 51 < ALT (IU/L) 87 ± ± 95 < ALP (IU/L) 129 ± ± 38 < rgt (IU/L) 526 ± ± 422 < TB (mg/dl) 2.6 ± ± 2.7 < Cholesterol (mg/dl) 147 ± ± PT (seconds) 12.3 ± ± CP score in patients with L/C 7.7 ± ± 2.0 < 0.001

14 Study 1 Characteristics of Patients Variable (mean ± SD) Probiotics group (n = 60) Placebo group (n = 57) Day 1 Day 7 p value Day 1 Day 7 p value Total protein (g/dl) 6.5 ± ± ± ± Albumin (g/dl) 3.5 ± ± ± ± AST (IU/L) 166 ± ± ± ± 62 <0.001 ALT (IU/L) 83 ± ± ± ± ALP (IU/L) 132 ± ± ± ± 27 <0.001 rgt (IU/L) 510 ± ± 430 < ± ± TB (mg/dl) 3.2 ± ± ± ± 0.7 <0.001 PT (seconds) 12.6 ± ± ± ± Cholesterol (mg/dl) 151 ± ± ± ±

15 Study 1 TNF-α Day 1 Day 7 IL1-b

16 Study 1 LPS Changes of microbiota after probiotics & placebo Variable (mean ± SD) Number of CFU per gram stool Probiotics group (n = 49) Placebo group (n = 35) Day 1 Day 7 p-value Day 1 Day 7 p-value Escherichia coli 404 ± ± ± ± Enterococci 341 ± ± ± ±

17 Study 2, Interim data Characteristics of Patients Variable (mean ± SD) Probiotics group (n = 50) Placebo group (n = 40) Day 1 Day 7 p value Day 1 Day 7 p value Total protein (g/dl) 6.8 ± ± ± ± Albumin (g/dl) 3.8 ± ± ± ± PT (seconds) 12.5 ± ± ± ± AST (IU/L) 188 ± ± ± ± ALT (IU/L) 113 ± ± ± ± rgt (IU/L) 432 ± ± ± ± TB (mg/dl) 1.9 ± ± 1.9 < ± ± TNF-α 79 ± ± ± ± IL ± ± ± ± LPS 1.13± ± 0.62 < ± ±

18 Study 2, Interim data Stool DGGE Line : type of microbiota Density: amount of microbiota No cirrhosis Probiotics group Day1 Day 7 Day1 Day 7 Cirrhosis Day1 Day 7 Day1 Day 7 Bacteroides coprocola unidentified Enterobacter sp. Enterobacter sp. Case 1 Case 2 Escherichia coli Enterobacter sp. Case 5 Case 6 At day 1, dominant type of bacteria was evident. The most dominant type of bacteria was G (-) bacteria. The density of G (-) bacteria (E. coli, Enterobacter) was decreased at day 7. Cirrhosis: dominant type of bacteria various different types of bacteria (diversity)

19 Study 2, Interim data Stool DGGE Placebo group No cirrhosis Cirrhosis Day1 Day 7 Day1 Day 7 Day1 Day 7 Day1 Day 7 Escherichia coli Enterobacter sp. Enterobacter sp. Case 9 Case 10 Case 13 Case 14 At day 1, dominant type of bacteria was evident. The most dominant type of bacteria was G (-) bacteria. The density of G (-) bacteria (Enterobacter, Bacteroides) was increased at day 7. Microbial distribution: dominant bacteria other dominant bacteria (simplification)

20 Study 2, Interim data One patient was treated by placebo and probiotics Case 16 Probiotics treatment is associated with change in gut microbiota.

21 Study 2, Interim data Summary of stool DGGE analysis (n=16) Dominant type of bacteria Day 1 Day 7 Change in distribution after Tx Probiotics No LC (n=4) Unidentified No Disappeared Different types of bacteria LC (n=4) E. coli Enterobacter No Disappeared Various different types of bacteria Placebo No LC (n=4) E. coli Enterobacter Dominant type of bacteria other dominant type of bacteria B LC (n=4) E. coli Bacteroides Enterobacter B Dominant type of bacteria other dominant type of bacteria

22 Summary Immediate abstinence is a most important treatment for patients with AH. Serum LPS was decreased by probiotics, especially, in patients with LC. In the stool culture, the number of CFU in E. coli was significantly decreased by probiotics. In the stool analysis, most dominant type of bacteria was G (-) bacteria in AH.

23 Summary In the stool analysis, probiotics treatment reduced the density of G (-) bacteria and placebo treatment increased the density of G (-) bacteria. Probiotics treatment changed microbial distribution from dominant type of G (-) bacteria to different types of bacteria. Placebo treatment simplified microbial distribution from dominant type of G (-) bacteria to other dominant type of G (-) bacteria.

24 Conclusion 7 days of oral supplementation with probiotics is associated with improvement of LPS and restoration of the bowel microbiota in AH.

25 Thank You For Attention!

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